1. Field of the Invention
This invention relates to a stable human oral cancer cell carcinoma cell line suitable for understanding the differences in the tumorigenic pathways implicated in the development and progression of oral squamous cell carcinoma obtained from the floor of the mouth of a chronic tobacco consumer.
2. Background of the Related Art
Oral cancer ranks as the sixth most common globally and is a major cause of cancer-related morbidity and mortality. The aetiology of betel and tobacco related oral cancer is considerably different to that resulting from smoking of tobacco. Exposure of the oral mucosa of habitual betel quid chewers to a plethora of carcinogenic constituents of tobacco and areca nut causes multiple genotoxic insults at the site bolus application, often resulting in the development of clinically distinct premalignant lesions, leukoplakia or erthroplakia, which undergo malignant transformation. Established human oral cancer cell lines are widely used to study the mechanism implicated in oral tumorigenesis. The human oral cancer cell lines available in Cell Repositories and Culture Collections around the world have been established from the Western or Japanese population and resulting from smoking of tobacco. In this respect, reference is made to Table 1.
Presently, there are no oral cancer cell lines resulting from chewing of tobacco. Majority of the studies on oral carcinogenesis have been carried out using tissue specimens (biopsy or surgically resected oral premalignant and malignant lesions) or cell lines resulting from smoking of tobacco. Majority of the studies on oral carcinogenesis has been carried out using tissue specimens (biopsy or surgically resected oral premalignant and malignant lesions) or cell lines resulting from smoking of tobacco. The recent awareness of inherited nature of some cancers, ethnic groups, existence of cancer families and importance of surveillance of high risk individuals using cancer susceptibility genes as markers emphasizes the need to establish oral cancer cell lines resulting from chewing of tobacco to provide a much needed model for oral tumorigenesis. The existing oral cancer cell lines are from tobacco smokers and thus are not suitable for studies pertaining to cancer susceptibility originating from chewing of tobacco. It may be argued that these studies could be carried in human oral cancer tissue specimens. However, in-depth studies carried out by the applicants have shown that the availability of the tissue specimen poses a major constraint on the work. Often the biopsy/FNAC specimens yield insufficient number of tumor cells for detailed molecular analysis. Furthermore, the yield of RNA from biopsy/surgically resected tissue specimens may be low reducing the feasibility of conducting studies aimed at identification of genes that are differentially expressed in different stages of oral tumorigenesis by Differential Display Reverse Transcription Polymerase Chain Research (DDRT-PCR). Hence, the non-availability of an experimental model system for tobacco induced oral cancer is a major obstacle in understanding the mechanism underlying oral tumorigenesis. Establishment of human oral cancer cell lines from betel and tobacco consumers is of utmost importance to provide an in vitro experimental model system for oral tumorigenesis.
An object of this invention is to propose a human oral cancer line established and propogated in vitro from the oral squamous cell curcinome obtained from the floor of mouth of a chronic tobacco consumer.
Another object of this invention is to propose a human oral squamous cell carcinoma cell line from the floor of the mouth of a habitual tobacco consumer for the study of genetic/molecular alterations involved in development and progression of an environmental carcinogen induced malignancy.
Still another object of this invention is to propose a human oral cancer line established and propagated in vitro from the oral squamous cell curcinome obtained from the floor of mouth of a chronic tobacco consumer for indentifying novel targets for use as diagnostic/prognostic markers and designing new therapeutic strategies for more effective management of cancer patients.
Yet another object of this invention is to propose a human oral cancer line established and propogated in vitro from the oral squamous cell carcinoma obtained from the floor of mouth of a chronic tobacco consumer which may be advantageously used for various applications as described hereinbelow.
According to this invention there is provided human oral cancer cell line established and propagated in vitro from oral squamous cell carcinoma obtained from the mouth of a chronic tobacco consumer, wherein said cell line AMOS-III has the following marker profile:
a. positive for tumor suppressor gene product, p53; marker of invasion and metastasis, ets-1; ternary complex factors, Net and elk; retinoic acid receptors, RXRxe2x88x9d; RARxe2x88x9d; anti-apoptotic protein and chaperone, HSP 70; epithelial specific antigen, ESA; human cytokeratin, CK 14, cell cycle regulatory protein, p21; Oncogene cyclin D1, heat shock protein, HSP90; transcription factor, ets-2; proliferation marker; Ki67.
b. Negative for human papilloma virus, HPV E6; mesenchymal cells marker, Vimentin; Low level of expression of oncogene MDM2, the p53 suppressor protein.
Further according to this invention there is provided a method of producing human oral cancer cell line which comprises:
a. subjecting oral squamous cell carcinoma from the floor of mouth to the step of biopsy in Hanks Balanced Salt Solution (HBSS) as a buffer supplemented with antibiotics (penicillin and streptomycin) and amphotericin B;
b. cutting the treated tissue of step (a) into smaller pieces;
c. washing the cut tissues with solution of antibiotics;
d. the washed tissues being introduced into tissue culture flasks having a medium comprising DMEM and Media 199 supplemented with fetal bovine serum (FBS) and growth supplements to allow the growth of cells comprising fibroblast cells which grow earlier than the epithelial cells.
e. Removing the fibroblasts cells from the culture to obtain a cell line comprising essentially of epithelial cells.